Carbohydrates are energy-sustaining biomolecules which are an essential part of our diet and are important in monitoring and treating diseases such as diabetes. Thus, sensitive and selective methods for the quantitative analysis of carbohydrates are required.
Liquid chromatography has been used effectively for the separation of carbohydrates in conjunction with several spectrophotometric detection techniques. Such detection methods include refractive index and post-column fluorometric detection methods. Refractive index detectors inherently exhibit poor sensitivity and fluorometric detectors often require extensive chemical alteration of carbohydrates, to include stronger chromophores in their chromophoric poor structures.
In addition to these detectors, electrochemical detectors have been developed for the quantitative analysis of carbohydrates. For example, Johnson et al. Anal. Chim. Acta 1983, 149 1-10 and Chim. Acta. 1981, 132 11-22 have developed a pulse amperometric detector (PAD) that allows for the quantitative analysis of sugars and other compounds in highly basic solutions following high performance liquid chromatographic separations. The mechanism of such analysis relies on the adsorption of carbohydrates on gold or platinum electrodes and their subsequent electrocatalytic oxidation. Triple-pulse waveform detectors have attracted considerable attention and have become commercially available, but exhibit the following drawbacks: 1) the pulse detection mode has a high charging current that results in poor sensitivity as compared to the commonly used DC detection mode; and 2) the formation of oxide layers on the electrode surface requires continuous pulsing (surface reactivation) to maintain activity, sensitivity and stability.
Nickel oxide electrodes have been employed for detection of sugars in alkaline solution. The detection limit has been reported to be 1 ng for monosaccharides and 5 ng for oligosaccharides. Santos and Baldwin Anal. Chem. 1987 59 1766-1770 used a carbon paste electrode modified with cobalt phthalocyanine (COPC), an electron-transfer mediator. The COPC chemically modified electrode (CME) has a limited lifetime and requires periodic pulsation (activation). A solution phase redox reaction of copper bis(phenanthroline) with various sugars allows sensitive detection of these compounds at -75 mV vs. Ag/AgCl. However, the reaction requires elevated temperatures.
A recent chemically modified electrode for carbohydrate detection was reported by Prabhu and Baldwin Anal. Chem. 1989 61 852-856 and Anal. Chem. 1989 61 2258-2263. They describe a glassy carbon electrode on which a layer of crystalline CuCl.sub.2 is deposited (a bare copper electrode). The lifetime of this electrode is 2-3 days, which is a marked improvement over COPC chemically modified electrode. The Cu-coated bare electrode offers enhanced sensitivity required for carbohydrate detection; however, the selectivity (aside from chromatographic resolution) and the long-term stability of such a catalytic electrode requires further improvement if it is to serve as a practical carbohydrate detector.
The present invention relates to a chemically modified electrode containing copper particles dispersed in a perfluorosulfonate ionomer sold under the trademark NAFION by du Pont. Polymer-coated chemically modified electrodes have been the subject of many on-going investigations. For instance polymeric films of polyaniline serve as anchoring (nucleation) sites for platinum. Such particles are three-dimensionally dispersed into the polymer matrix. The advantages accrued from this type of electrode are mechanical stability and improved selectivity, which can be achieved by the controlled electrodeposition of metal into the polymer.
Films made of perfluorosulfonate ionomers, are quite inert in most electrolytes and adhere very strongly to substrates. Perfluorosulfonate ionomers have been used extensively as permselective coatings, sensors for incorporating cations, and charge exclusion membranes. The present inventors have found that a perfluorosulfonate ionomer deposited on glassy carbon and having copper and copper oxides electrolytically deposited into the ionomer is an electrode which can be successfully used as a working cell in a liquid chromatograph detector in qualitative analysis and quantitative analysis of carbohydrates.